Strand Conformation Research Articles

The sequence TGAAKAVALVL from glyceraldehyde‐3‐phosphate dehydrogenase displays structural ambivalence and interconverts between α‐helical and β‐hairpin conformations mediated by collapsed conformational states

The peptide TGAAKAVALVL from glyceraldehyde-3-phosphate dehydrogenase adopts a helical conformation in the crystal structure and is a site for two hydrated helical segments, which are thought to be helical folding intermediates. Overlapping sequences of four to five residues from the peptide, sample both helical and strand conformations in known protein structures, which are dissimilar to glyceraldehyde-3-phosphate dehydrogenase suggesting that the peptide may have a structural ambivalence. Molecular dynamics simulations of the peptide sequence performed for a total simulation time of 1.2 micros, starting from the various initial conformations using GROMOS96 force field under NVT conditions, show that the peptide samples a large number of conformational forms with transitions from alpha-helix to beta-hairpin and vice versa. The peptide, therefore, displays a structural ambivalence. The mechanism from alpha-helix to beta-hairpin transition and vice versa reveals that the compact bends and turns conformational forms mediate such conformational transitions. These compact structures including helices and hairpins have similar hydrophobic radius of gyration (Rgh) values suggesting that similar hydrophobic interactions govern these conformational forms. The distribution of conformational energies is Gaussian with helix sampling lowest energy followed by the hairpins and coil. The lowest potential energy of the full helix may enable the peptide to take up helical conformation in the crystal structure of the glyceraldehyde-3-phosphate dehydrogenase, even though the peptide has a preference for hairpin too. The relevance of folding and unfolding events observed in our simulations to hydrophobic collapse model of protein folding are discussed.

Conformational Diversity of the Fibrillogenic Fusion Peptide B18 in Different Environments from Molecular Dynamics Simulations

The development of specific agents against amyloidoses requires an understanding of the conformational behavior of fibrillogenic peptides in different environments on the microscopic level. We present extensive molecular dynamics simulations of the fibrillogenic Bindin (103-120) B18 fusion peptide for several different environments: a water-trifluorethanol (TFE) mixture, pure water, aqueous buffer containing 100 mM NaCl, and a buffer-vapor interface. The peptide was studied as an isolated molecule in solution or at an interface. In the simulations, the conformational behavior of the peptide was found to strongly depend on the environment in agreement with experimental data. Overall, large portions of the peptide were unstructured. Preformed alpha-helical conformations were least stable in pure water and most stable in the water-TFE mixture and the buffer-vapor interface. In all environments, the alpha-helical conformation was most stable in the region around residues 113-116, which are mainly hydrophilic. Extended configurations in water or buffer folded into structures containing beta-sheets in agreement with data from circular dichroism spectroscopy. In buffer, the beta-sheet content was larger than in water and alpha-beta transitions were observed at elevated temperature. Beta-sheets were formed by hydrophobic residues; turns were formed by hydrophilic residues. A few typical beta-sheets that contain different residues are suggested. A B18 molecule in a strand-loop-strand conformation placed in buffer in contact with vapor was spontaneously adsorbed to the buffer-vapor interface with its hydrophobic side pointing toward the vapor phase. The adsorption induced the formation of turns at positions 108-119 and alpha-helical conformations in the region around residues 114-117. Alpha-helices were parallel to the interface plane in agreement with data from IR reflection absorption spectroscopy.

Investigations on C–H⋯π interactions in RNA binding proteins

We have investigated the roles played by C–H⋯π interactions in RNA binding proteins. There was an average of 55 C–H⋯π interactions per protein and also there was an average of one significant C–H⋯π interaction for every nine residues in the 59 RNA binding proteins studied. Main-chain to side-chain C–H⋯π interactions is the predominant type of interactions in RNA binding proteins. The donor atom contribution to C–H⋯π interactions was mainly from Phe, Tyr, Trp, Pro, Gly, Lys, His and Ala residues. The acceptor atom contribution to main-chain to side-chain C–H⋯π and side-chain to side-chain C–H⋯π interactions was mainly from Phe and Tyr residues. On the contrary, the acceptor atoms of Trp residues contributed to all the four types of C–H⋯π interactions. Also, Trp contributed both donor and acceptor atoms in main-chain to side-chain, main-chain to side-chain five-member aromatic ring and side-chain to side-chain C–H⋯π interactions. The secondary structure preference analysis of C–H⋯π interacting residues showed that, Arg, Gln, Glu, His, Ile, Leu, Lys, Met, Phe and Tyr preferred to be in helix, while Ala, Asp, Cys, Gly, Trp and Val preferred to be in strand conformation. Long-range C–H⋯π interactions are the predominant type of interactions in RNA binding proteins. More than 50% of C–H⋯π interacting residues had a higher conservation score. Significant percentage of C–H⋯π interacting residues had one or more stabilization centers. Seven percent of the theoretically predicted stabilizing residues were also involved in C–H⋯π interactions and hence these residues may also contribute additional stability to RNA binding proteins.

Cloning and characterization of scale β‐keratins in the differentiating epidermis of geckoes show they are glycine‐proline‐serine–rich proteins with a central motif homologous to avian β‐keratins

The beta-keratins constitute the hard epidermis and adhesive setae of gecko lizards. Nucleotide and amino acid sequences of beta-keratins in epidermis of gecko lizards were cloned from mRNAs. Specific oligonucleotides were used to amplify by 3'- and 5'-rapid amplification of cDNA ends analyses five specific gecko beta-keratin cDNA sequences. The cDNA coding sequences encoded putative glycine-proline-serine-rich proteins of 16.8-18 kDa containing 169-191 amino acids, especially 17.8-23% glycine, 8.4-14.8% proline, 14.2-18.1% serine. Glycine-rich repeats are localized toward the initial and end regions of the protein, while a central region, rich in proline, has a strand conformation (beta-pleated fold) likely responsible for the formation of beta-keratin filaments. It shows high homology with a core region of other lizard keratins, avian scale, and feather keratins. Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis show a higher beta-keratin gene expression in regenerating epidermis compared with normal epidermis. In situ hybridization confirms that mRNAs for these proteins are expressed in cells of the differentiating oberhautchen cells and beta-cells. Expression in adhesive setae of climbing lamellae was shown by RT-PCR. Southern blotting analysis revealed that the proteins are encoded by a multigene family. PCR analysis showed that the genes are presumably located in tandem along the DNA and are transcribed from the same DNA strand like in avian beta-keratins.

Probabilistic Approach to Determining Unbiased Random-coil Carbon-13 Chemical Shift Values from the Protein Chemical Shift Database

We describe a probabilistic model for deriving, from the database of assigned chemical shifts, a set of random coil chemical shift values that are "unbiased" insofar as contributions from detectable secondary structure have been minimized (RCCSu). We have used this approach to derive a set of RCCSu values for 13Calpha and 13Cbeta for 17 of the 20 standard amino acid residue types by taking advantage of the known opposite conformational dependence of these parameters. We present a second probabilistic approach that utilizes the maximum entropy principle to analyze the database of 13Calpha and 13Cbeta chemical shifts considered separately; this approach yielded a second set of random coil chemical shifts (RCCSmax-ent). Both new approaches analyze the chemical shift database without reference to known structure. Prior approaches have used either the chemical shifts of small peptides assumed to model the random coil state (RCCSpeptide) or statistical analysis of chemical shifts associated with structure not in helical or strand conformation (RCCSstruct-stat). We show that the RCCSmax-ent values are strikingly similar to published RCCSpeptide and RCCSstruct-stat values. By contrast, the RCCSu values differ significantly from both published types of random coil chemical shift values. The differences (RCCSpeptide - RCCSu) for individual residue types show a correlation with known intrinsic conformational propensities. These results suggest that random coil chemical shift values from both prior approaches are biased by conformational preferences. RCCSu values appear to be consistent with the current concept of the "random coil" as the state in which the geometry of the polypeptide ensemble samples the allowed region of (phi, psi)-space in the absence of any dominant stabilizing interactions and thus represent an improved basis for the detection of secondary structure. Coupled with the growing database of chemical shifts, this probabilistic approach makes it possible to refine relationships among chemical shifts, their conformational propensities, and their dependence on pH, temperature, or neighboring residue type.

Nearest‐neighbor effects and structural preferences in dipeptides are a function of the electronic properties of amino acid side‐chains

The electronic properties of amino acid side-chains are emerging as an important factor in the preference for secondary structure in proteins. These properties have not been fully characterized, nor has their role in the behavior of peptides been explored in any detail. The present studies sought to evaluate several possibilities: 1) that hydrophilicity can be expressed solely in electronic terms, 2) that substituent effects of side-chains extend across the peptide bond, and (3) nearest-neighbor effects in dipeptides correlate with secondary structural preferences. Quantum mechanics (QM) calculations were used to define the electronic properties of individual amino acids and dipeptides. It was found that the hydrophilicity of an amino acid side-chain can be accurately represented as a function of the electron densities of its component atoms. In addition, the nature of an amino acid in the second position of a dipeptide affects the electronic properties (Mulliken populations and electron densities) of the main-chain atoms of the first residue. Certain electronic features of the dipeptides strongly correlated with propensity for secondary structure. Specifically, Mulliken population data at the Calpha atom and N atom predicted preference for alpha-helices versus coil and strand conformations, respectively. Analysis of dipeptides arrayed in either helical or extended structures revealed lengthening of main-chain bonds in the alpha-helical conformations. A thorough characterization of the electronic properties of amino acids and short peptide segments may provide a better understanding of the forces that determine secondary structure in proteins.

Enhancement and Inhibition by 2′-O-Hydroxyethyl Residues of Gene Targeting Mediated by Triple Helix Forming Oligonucleotides

Reagents that recognize and bind specific genomic sequences in living mammalian cells would have great potential for genetic manipulation, including gene knockout, strain construction, and gene therapy. Triple helix forming oligonucleotides (TFOs) bind specific sequences via the major groove, but pyrimidine motif TFOs are limited by their poor activity under physiological conditions. Base and sugar analogues that overcome many of these limitations have been described. In particular, 2′-O-modifications influence sugar pucker and third strand conformation, and have been important to the development of bioactive TFOs. Here we have analyzed the impact of 2′-O-hydroxyethyl (2′-HE) substitutions, in combination with other 2′ modifications. We prepared modified TFOs conjugated to psoralen and measured targeting activity in a gene knockout assay in cultured hamster cells. We find that 2′-HE residues enhance the bioactivity of TFOs containing 2′-O-methyl (2′-OMe) modifications, but reduce the bioactivity of TFOs containing, in addition, 2′-O-aminoethyl (2′-AE) residues.

Self-assembly and structure transformations in living polymers forming fibrils

The classical isodesmic one-dimensional model for equilibrium polymerization is generalized in order to describe self-assembly in systems forming fibrils. The model was applied to peptide solutions forming -sheet tapes which can further aggregate into stacks of various thickness: double tapes and fibrils (several double tapes stacked together). We found that in some cases the model yields several step-like transitions as the concentration increases: first from monomers to single or double tapes, and then to fibrils. The abruptness of the first transition is controlled by the free energy penalty for transformation of a peptide from random coil to a straight -strand conformation (the latter is characteristic for tapes). If both single and double tapes are allowed, the length of the aggregates after the first transition can be very large with high scission energies. For very low energies of attraction between double tapes, the transition from double tapes to fibrils happens separately (above the first transition), and it is even more abrupt and produces extremely long fibrils. The theoretical findings are used to extract the characteristic molecular parameters for the self-assembly of the de novo peptide DN1 forming polymeric -sheets in water.

Conformational properties of DNA dodecamers containing four tandem repeats of the CNG triplets.

We studied DNA dodecamers (CAG)4, (CCG)4, (CGG)4 and (CTG)4by CD spectroscopy and polyacrylamide gel electrophoresis. Each dodecamer adopted several ordered conformers which denatured in a cooperative way. Stability of the conformers depended on the dodecamer concentration, ionic strength, temperature and pH. The dodecamers, having a pyrimidine base in the triplet center, generated foldbacks at low ionic strength whose stem conformations were governed by the GC pairs. At high salt, (CCG)4 isomerized into a peculiar association of two strands. The association was also promoted by high oligonucleotide concentrations. No similar behavior was exhibited by (CTG)4. At low salt, (CGG)4 coexisted in two bimolecular conformers whose populations were strongly dependent on the ionic strength. In addition, (CGG)4 associated into a tetraplex at acidic pH. A tetraplex was even observed at neutral pH if the (CGG)4 concentration was sufficiently high. (CAG)4 was very stable in a monomolecular conformer similar to the known extremely stable foldback of the (GCGAAGC) heptamer. Nevertheless, even this very stable conformer disappeared if (CTG)4 was added to the solution of (CAG)4. Association of the complementary strands was also strongly preferred to the particular strand conformations by the other couple, (CCG)4 and (CGG)4.

Preferences of the side chains in proteins for helix, beta strand, turn, and other conformations. Secondary structures of copper proteins

Statistical preferences of the side chains in proteins for the helical conformation, the β strand conformation, and the turn conformation were derived from the computer-assigned secondary structures of 55 protein chains reported by Levitt and Greer in 1977 after averaging redundant structures and weighting structures that showed > 25 % homology by w = ▪. Separate preferences are reported for free cysteine side chains and for cystine. These preferences are used to predict the secondary structures of cucumber plastocyanin and cupredoxin, whose crystal structures will be reported soon. The loops in the C-terminal copper-binding regions are discussed.

Magnesium Cation Induced Conformational Change of Yeast tRNAPhe as Studied by Singlet-Singlet Energy Transfer

The A76 or A73 nucleotide at the 3'end of tRNAPhe was modified with the fluorescent reagent of proflavine (PF). The distance between the fluorophore of the 3'end and the Y base was measured by singlet-singlet energy transfer under the conditions of 10 mM and 0.01 mM Mg2+. The distance obtained at 10 mM Mg2+ is very close to that obtained by the X-ray diffraction method, while the distance at 0.01 mM Mg2+ is significantly smaller. The difference in the distance is explained as a result of destabilization of the tertiary structure with reduction of the Mg2+ concentration. The calculated distance between A73 and A76 shows the stacked conformation of the CCA strand. Fluorescent quenching experiments showed that the degree of stabilization of 3'end A76 by stacking is lower than that of A73. The removal of the CCA segment causes a difference not only in the thermal melting curves but also in the fluorescent wavelength of the Y base at 0.01 mM Mg2+. The results suggest that the 3'end CCA strand has a helical structure and contributes to the stabilization of the whole structure of tRNAPhe.